HIGH-DENSITY-LIPOPROTEIN SUBFRACTION-3 INTERACTION WITH GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEINS

To elucidate further the binding of high-density-lipoprotein subfracti on 3 (HDL3) to cells, the involvement of glycosylphosphatidylinositol- anchored proteins (GPI-proteins) was studied. Treatment of cultured ce lls, such as fibroblasts or SK-MES-1 cells, with a phosphatidylinosito l-specific phospholipase C (PI-PLC) significantly decreases specific H DL3 binding. Moreover, PI-PLC treatment of cultured cells or cellular plasma membrane fractions results in releasing proteins. These protein s have a soluble form and can also bind HDL3, as revealed by ligand bl otting experiments with HDL3. In order to obtain enriched GPI-proteins , we used a detergent-free purification method to prepare a caveolar m embrane fraction. In the caveolar fraction, we obtained, by ligand blo tting experiments, the enrichment of two HDL3-binding proteins with mo lecular masses of 120 and 80 kDa. These proteins were also revealed in a plasma membrane preparation with two other proteins, with molecular masses of 150 and 104 kDa, and were sensitive to PI-PLC treatment. El ectron microscopy also showed the binding of Au-labelled HDL3 inside t he caveolar membrane invaginations. In SK-MES-1 cells, HDL3 are intern alized into a particular structure, resulting in the accumulation and concentration of such specific membrane domains. To sum up, a demonstr ation has been made of the implication of GPI-proteins as well as cave olae in the binding of HDL3 to cells.